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Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5'-flanking region.

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0022-538X/91/031558-10$02.00/0

Copyright X3 1991, American Society for Microbiology

Variable Expression of

Latent

Membrane Protein in Nasopharyngeal

Carcinoma Can Be Related

to

Methylation Status of the

Epstein-Barr Virus

BNLF-1

5'-Flanking

Region

HU LI-FU,tJANOS MINAROVITS,T CAO SHI-LONG,§ BERTHA CONTRERAS-SALAZAR, LARS RYMO,II KERSTIN FALK, GEORGE KLEIN, ANDINGEMAR ERNBERG*

DepartmentofTumorBiology, Karolinska Institute, Box60400, 10401 Stockholm, Sweden

Received 8August1990/Accepted 6 December 1990

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In

nasopharyngeal carcinoma(NPC),only EBNA-1 is regularlyexpressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patternsoftheEBV genome varied between these cell types. Invirallytransformedlymphoblastoid

cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's

lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1(inboth tumortypes)and LMP(inC15).Twopresumptivecontrolregionswereexemptedfrom this overall methylation: the oriP that contains both an origin ofDNA replication and an EBNA-1-dependent enhancer and the5'-flanking regionof the BNLF-1 openreadingframe that codes for LMP. The latter wasonly

exemptedin the LMPexpressing NPC.We have nowinvestigatedthe relation betweenexpressionof LMP and

methylation ofDNA in the5'-flanking 1kbregion of BNLF-1,coding forLMP. LMPwasmethylatedin3 of 12NPC biopsiesthat did not express LMP but waspartiallyortotallyunmethylatedin theremaining9 that

expressedtheprotein. The three BNLF-1 exons werehighly methylatedin allthe tumors. The oriPregionwas unmethylatedin all the tumors, as in thepreviously studiedRael cell line and nudemouse-passagedNPC.Also, theBamHIWenhancerregion involvedin theexpression ofEBNAnuclearproteinswasmethylated. Noneof

thebiopsies expressedEBNA-2. Ourdata show that theEBVgenomesarehighlymethylated inNPCtumors. The strong reversecorrelationbetweenthemethylationof theputativecontrolregionof the LMP gene and the

expression of LMP suggests thatmethylationhas a role in theregulation ofthis gene.

Seven virus-coded proteins are regularly expressed in Epstein-Barr virus (EBV) transformed lymphoblastoid cell

lines(LCLs): the nuclear proteins EBNA-1 toEBNA-6and thelatent membrane protein (LMP) (8, 9,18-21, 24, 30, 32). In nasopharyngeal carcinoma (NPC) only EBNA-1 and

LMP were detected (14, 43), and in Burkitt's lymphoma (BL) tumors and phenotypically representative cell lines

onlyEBNA-1 wasdetected (13,35, 36). Sofar, EBNA-1 has been reported in 75 NPC biopsies, whereas LMP was only detected in 45 of 80 (56%) NPC biopsies. The biopsies showed similar expression patterns, irrespective of

geo-graphical origin (North andEastAfrica, China, Malaysia,or

the United Kingdom), although more of the Chinese NPC

biopsies (71%)thanthoseof EastorNorthAfrican origin (25

to 62% in the two published studies) expressed LMP.

EBNA-2 to EBNA-6 could not be detected in any of the

biopsies.

Wehaverecently shown that the

methylation

of theEBV

genomes differs among the three EBV-carrying cellular

* Correspondingauthor.

t Permanent address: Shanghai Cancer Institute, Shanghai

200032,People's Republic of China.

t Permanent address: Microbiology Research Groupof the Na-tionalInstitute of Hygiene, Budapest, Hungary.

§ Permanent address: ShanghaiMedical University Tumor Hos-pital, Shanghai,People's Republic of China.

brPresentaddress:DepartmentofMedicalBiochemistry,

Gothen-burg University, 40033 Gothenburg,Sweden.

prototypes. InEBV-transformed B-cell lines ofnormal

ori-gin (LCLs), the viralgenomes areunmethylated (12). In the

BL-derived cell line Rael and in the nude mouse-passaged

NPC C15 tumor, CpG pairs of the EBV genomes were

extensively methylated. The exons of expressed genes,

EBNA-1 and LMP, were methylated as well, but two

potential control regions were unmethylated: the oriP that

contains one origin of DNA replication and an EBNA-1-dependent andEBNA-regulating enhancer (31, 38) and the

5'-flanking regionofBNLF-1thatcodes for theLMP(inthe

NPC, which expressed LMP). Rescue of the virus from BL andfrom NPC into LCLs has led to demethylation of the viral DNA in allderived lines.

DNAmethylationcanparticipatein the controlof

eukary-oticgenes(6, 10). The cause-effectrelationshipis

controver-sial (6, 11, 23). Recent studies have shown that the methyl-ation ofspecific CpG pairswithin theDNA-binding motifsof transcription factorscanpreventtheir binding and downreg-ulate thetranscriptionofanadjacentgene(2,28, 41).Hence,

the methylation ofspecific sitescan directly influencegene

expression and hypomethylation of 5' promoter-enhancer

regions is often correlated with expression, whereas

meth-ylation of structuralgenesis not(6).

The cell phenotype-dependent differences in EBV DNA

methylation and the apparent correlation with viral gene

expressionsuggestthatmethylation is involved in viralgene

control in latently infected cells (12). This suggestion was

tested in thepresent paperby focusingonthefact that only about56% of NPCsexpressLMP(14). Wehaveinvestigated 1558

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the methylation of CpGs in BNLF-1 and in 5'-flanking

regions. The putative EBNA-controlling region in oriPand the enhancer in the internal repeat 1 have been studied in parallel. We have found a strongcorrelation between LMP expression and lackof methylation of the 5'-flanking region ofBNLF-1, extending1 kbupstreamof the promoter. This correlation is in line withtherecently discovered regulating

elements in this region involved in LMP control (12a, 17). The correlation between LMP expression and the

methyla-tion status of the LMP regulatory sequence suggests a

modulating role of methylation or, alternatively, that the

methylation status depends on the activity of the LMP

regulatorysequence.

MATERIALS AND METHODS

Cell lines and tissue culture. Rael isaBL-derived cell line

(27) with a stable type I BL phenotype, expressing only

EBNA-1 but not EBNA-2 to -6 or LMP. The CBC- and

NAD-C15-STO cell lineswereestablishedby transfer of the

virus carried by the NPC designated C15, serially

propa-gated in nude mice, to normal B cells (12). All lines were

grownin RPMI 1640 medium(GIBCO Biocult,Long Island,

N.Y.), supplemented with 100 U ofVpenicillin perml, 100

,ug of streptomycin per ml, and 10% fetal calfserum. The

cells werefed every 3 to4 days and cultured in incubators with 5% CO2 atmosphere andhigh humidity.

Biopsies. NPC biopsies were doneby local surgery in the

Shanghaiarea. The tumorswerestored frozen (-70°C) until

prepared for DNA or protein blotting. CAO is a Chinese

EBV DNA-positive NPC tumorpassaged in nude mice (20

passages [5]). C15, C17,andC18 arenudemouse-passaged, EBVDNA-positive African NPC tumors, analyzed after 15 to 50passages (4). The tissues were prepared for

immuno-blottingas describedearlier(14).

Immunoblotting. Immunoblotting was performed as

de-scribed earlier (13, 14). A previously characterized serum

samplefromahealthy EBV-seropositive donorwasusedas the antibody source fordetection of EBNAs (VCA, 1:320; EAD, <1:10; EAR, 1:320; EBNA, 1:320; and EBNA-2, 1:80). Alternatively, wealso used the seraofaCLLpatient PG (VCA, 1:1,280; EA(D), 1:640; EA(R), 1:20; EBNA-1, 1:320; and EBNA-2, 1:80). EBNA-1 was detected by a humanserumthat has beenaffinitypurified on an

EBNA-1-specific polypeptide (no. 107) (14). LMPwasdetectedbythe

S-12 monoclonal antibody (MAb) provided by D.

Thorley-Lawson(DepartmentofPathology, TuftsUniversity School ofMedicine, Boston, Mass.). The humanserumsamplewas used at a 1:20 dilution and the S-12 MAb was used at a

1:1,000 dilution of ascitic fluid. Alternatively, a pool of MAbsagainst LMP, kindly suppliedby Martin Rowe(CRC Laboratories, Birmingham, United Kingdom; CS.1 to CS.4

[34]), wasusedata1:1,000dilution. LMP could be detected in a control sample prepared from B95-8 cells when an

immunoblotting lane was loaded with material

correspond-ingto 5 x 103 cells. LMP could not bedetected in samples

withlower numbersof cells. Normallythe lanesare loaded

with material from 105 to 106 cells; LMP would thus be detected if it were expressed in 0.5% of the cells (given a level ofexpression per cell similar to that of B95-8 cells).

This givesan indication of the levelofsensitivity needed to detect LMP in NPC biopsies, although accurate cell num-bers inbiopsies cannotbe determined.

Analysis of DNA methylation. Eukaryotic and viral DNA

are predominantly methylated in CpG pairs. Methylation

was analyzed by digesting high-molecular-weight cellular

m021

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T

916

T

- I . I

b.

C.

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__ilU

11

ill557r

1llllllllllllllmlllm,!__

d.

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1132 III ,, ,,,, in,529,

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e.

11671304 -§67i It494 ,,

f.

357, 818 ,

L.6

169424

506 ,,iA.

1722

,,213,, nl .i,i1

FIG. 1. DNA probes used to study methylation by Southern blotting. Horizontalblacklines(_)between boxeswithnumbers (e.g.,7032)showprobes usedinthisstudy.Inseveral casessmaller probes(t)wereused as an alternative. Small vertical barsindicate restriction sites detectedby theisoschizomerMspIorHpaII (in the latter caseonlywhen theCpGwasunmethylated). Major fragments resulting fromcompletedigestion withthese enzymes areindicated by size (in base pairs). Identified enhancers and EBNA-1-binding regions have been indicated within the probes (IZ1). The major latent promoters (

r)

and the early promoterfor the latent

mem-brane protein

(PI)

areindicated. oriP and the exonsforEBNA-1, EBNA-2,and LMP areindicated beneath theprobes (L ).(a)oriP region in theBamHI C fragment. In particularthe916-bpfragment within oriP, cleaved out by MspI (t) was used. (b) BamHI W fragment, with theAvaIfragmentusedas a probetodetectthe W enhancer (t). The figure shows the first of a series of identical BamHI W fragments. (c) Probe used to detect the 3' part ofthe EBNA-2-coding exon. (d) BamHI K fragment, with a smaller

Mspl-MspI probewithin the repeatregionof BKRF-1(t). (e) Probe recognizingthe LT 3region,obtainedby digestion oftheEcoRIDhet fragment with XhoI. A, B,andC showthethreeexonsforLMP.(f) Twoprobes foranalysis ofthe5'-flankingsequenceofLT3, either as anXhoI-EcoRl fragmentorobtainedbydigestionwithBgIl (t). The maps andcoordinatesgivenare basedonthe B95-8 sequence and onmolecular weight markers included in our blots (HindIII-cleaved lambda DNA anda1-kb ladder(BethesdaResearch Labo-ratories).

and viral DNAs with either HpaII (specificity, CCGG) or

HhaI(specificity, GCGC). For each 1 pgofDNA, 10 U of

enzymewasaddedtocompletedigestion. TheHpaIIdigests were controlledbyparallel digestion withMspI, an isoschi-zomerwhich isnotinhibitedby

methylation.

The restriction

enzymes were obtained from Amersham

(Amersham,

En-gland). Digestion was performed

overnight

or for longer

times in the

enzyme-specific

buffers

provided by

the manu-facturer.

9 I F4-99-6-4.

1689

.--T

774

Till

lt.c. 1101

:d

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r CM CO) f L) to r~- o O7)rYO r-v

00

N ( 0)

rrv-m

C

116.

-

#, .

97

-68

-...

-EBNA- I

FIG. 2. Immunoblots showing expression ofEBVproteinsinNPCbiopsies. (A) Detection ofLMPwithS-12 MAbs.Biopsies 3, 5, and 10 werenegative, while expressionwasweak butpositivein 7, 12,and 13. (B) Detection ofEBNA-1 byamonospecific human EBNA-1 serum,obtainedbyaffinity purificationwithanEBNA-1polypeptide.(C) Detection ofEBNAswithapolyvalentEBNA-positiveserum(PG).

Ramos wasused as anegative control, and B95-8 was used as apositivecontrol. Thepositionsof standard molecularweightmarkersaswell asthe positions of EBNA-1 and LMP are indicated.

ThedigestedDNAswereseparatedon a1.5% agarose gel

(Bethesda Research Laboratories, Inc., Bethesda, Md.) in

Tris-borate-EDTA buffer (TBE; 89 mM Tris base, 89 mM

boricacid, 2 mM EDTA). Electrophoresis was carried out

for 13hat50 V.Digestionwaschecked by ethidium bromide

staining. Denaturationwascarried out in 0.5 M NaOH with

1.5 MNaCifor1h.The DNA was then neutralized for 1 h in

1 M Tris-1.5 M NaCl, pH 7.5. Transfer to nitrocellulose

(HybondN;Amersham) was done in 20x SSPE (Ix SSPE is

0.18 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA

[pH 7.7]) overnight. Prehybridization was performed with

6x SSPE-1% sodium dodecyl sulfate (SDS)-100 p.g of

herring sperm DNA (Boehringer Mannheim) per ml-lOx

Denhardt solution(0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serumalbumin) at42°C overnight.

Theprobes were labeled with[32P]dCTP (Amersham) by

random primer oligolabeling with the Klenow fragment of Escherichia coli DNA polymerase (16). Hybridization with

specific probes was performed at 42°Cfor 16 to 24 hin 6x

SSPE-100 ,ug of herring sperm DNA-50% formamide-1%

A

CA

0

E

(U

116 -97.

66

-43.

a

a

.4

.A_

B

- LMP

116-97

-66--!F

ip

lEBNA-I

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TABLE 1. Designations and EBV protein expression in biopsies and cell lines used inthis study

Expression

Cell(origin)a Reference

EBNA-1 EBNA-2to -6 LMP

1(ChNPC) + - +

2(ChNPC) + - +

3(Ch NPC) + -

-4(Ch NPC) + - +

5(Ch NPC) + -

-6(Ch NPC) + - +

7(Ch NPC) + - +

8(Ch NPC) + - +

9(Ch NPC) + - +

10 (Ch NPC) + -

-11(Ch NPC) + - +

12(Ch NPC) + - +

13(ChNPC) + - +

C15(NuMo NPC) + - + 4

CAO (NuMo NPC) + - + 5

B95-8 (MarmosetLCL) + + +

Rael(BL) + - - 29

CBM1-Ral-STO(LCL) + + + 12

CBC-SEB-CHI-3 (LCL) + + +

CBC-SEB-CHI-7 (LCL) + + +

a Ch, Chinese; NuMo, nude mousepassaged; B95-8, marmoset B cells

transformed by infectious mononucleosis-derived EBV;CBC, cord

blood-derived cells. Rael and CBM1-Ral-STO carrythe samestrainof EBV.

C) Q

wU LuI

04 V) m m

Ut) C

msM U ('_T-Rt T- o0*

0

M H MH M H M H M H M H M HM H M H

4,0-2,0

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*

*

1,0-0 aS

a 0

0,50- a

0,39- _ . _ S

0,34

-0,30- - * * 0

dextran sulfate containing the radioactive probe. Subse-quently the filters were washed in 2xSSPE-0.1% SDS for15 min at room temperature, two times in 0.2x SSPE-0.1% SDS for30 min at 65°C, and finally for 5 min in 0.lx SSPE. The filters werefinally exposed to Fuji medical X-ray film for 4 to48 h at 90°C and then developed (12).

Probes. The following probes were used (Fig. 1): the BamHI K fragment; the pMBamH2 fragment (cloned from the M-ABAcell line, kindly provided by Georg Bornkamm), which covers the 3' part of the EBNA-2A exon BYRF-1 (coordinates 48848 to 49964); a MspI-MspI digest from the BamHICfragment(coordinates 8189 to 9105), coveringpart

of oriP;aMspI-MspIfragmentfrom the BamHI K fragment, covering part of the EBNA-1 exon BKRF-1 (coordinates 108161 to 108924); an XhoI-XhoI fragment from the EcoRI

Dhet

fragment covering most of the LMP exons (coordinates 167484 to 169424); an XhoI-EcoRI fragment (coordinates 169424 to 172281) and a BglI-BglI fragment (coordinates 169449 to 170290), both covering the promoter and

5'-flanking sequences of LMP; and an AvaI-AvaI fragment

covering the BamHI W enhancer (coordinates 13827 to

14460).

RESULTS

Expression of EBV-encoded proteins. Thirteen Chinese

NPC biopsies fromtheShanghai area were included in this

study. They were EBV DNA positive, as shown by Southern blotting (for details, see below). Expression of the EBNAs and LMP was assayed by immunoblotting by using the polyvalent human serum, PG (Fig. 2C), the affinity-purified

O ul z

4 . o X

o) 0 co n _ a:

M H M H M H M H M H M H

it

4D

d

i

_,_A *

*

-0

FIG. 3. Methylationpatternofthe BNLF-1exonsinNPCbiopsies.Biopsyandcontrol cell line DNAscleaved withHpaII(H)orMspI

(M) and probed with an XhoI-XhoI probe (Fig. le). Biopsies 3, 5, and 10 were LMP negative. As controls, B95-8 (predominantly

unmethylated), Rael(methylated), andtwoEBV-transformedLCLs(CBC-SEB-CHI-3and-7,unmethylated)wereused. Thepositionsof the molecularweightmarkerfragmentsareindicated.TheeasilyidentifiedmajorB95-8fragmentscanalsobe usedasreferences(357,494, and 818bp).

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w W- N q'

M H M H MH MH

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0

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FIG. 4. Methylation pattern of the 5' BNLF-1-flanking region. Same blot shown Fig. 3, strippedandreprobedwithaBgIl-BgIl probe

(smallersubfragmentinFig. if),coveringthe promoter andapproximately800bp upstreamoftheLMPexons.SampleDNAs cleaved with

HpaII and MspI. Biopsies 3, 5, and 10 and the control cellline Rael didnotexpressLMP,while all the othersamplesdid. Thepositionsof

the DNAmolecularweight markerfragmentsaswellasthepositionsof three minor B95-8fragments (in parentheses: 177,146,and 107bp; theyare easytoidentify togetherwith thelarger 506-bpfragment)areindicated.

(a) B95-8

5' 3

(b) NPC-12

LT

...

t ttt

t

Msp I

LT

...

tttT

Hpa II

(c)

NPC-10

MspI

Hpa II

0.1 kb

FIG. 5. Schematic drawingof theBNLF-1 region andupstream sequences in twoNPCbiopsies(oneexpressing LMP [b; no. 12] and one notexpressingLMP[c; no. 10]). MspIandHpaIIcleavage sites

areindicated(O).Forreference,the B95-8 map withcleavage sites, LMP exons, andthepromoter isprovided (a).

human serum specificfor EBNA-1 (Fig. 2B), and the S-12

MAbfor LMP(Fig. 2A).The dataonprotein expressionare summarized in Table 1 and are related to those of earlier studies summarized in Table 2. All EBV DNA-positive biopsies expressed EBNA-1, as shownby both the

polyva-lent and themonospecificreagent.Ten of the thirteen(77%)

EBVDNA-positive biopsieswereLMPpositive (no. 1, 2, 4,

6, 7, 8, 9, 11, 12, and 13). Ofthese, no. 7, 12, and 13 gave

relatively weakersignals. Nevertheless, inspectionof these lanes in reflected lightandbytransillumination showed that

theywereclearly positive,whereasnosignalatallcould be detected in no. 3, 5, and 10. Biopsy 7 hada slightly higher LMPmolecularweightthan the others. A small variation in LMP size is common. C15 and CAO have been shown in earlier studies tobe LMPpositive (4, 14).

Methylation of the BNLF-1 readingframes (LMP coding)

and the 5'-flanking region. Twelve of the biopsies were selected for DNA methylation studies. Two nude

mouse-passaged NPC tumors, C15 (4) and CAO (5), were also included. As controls we have included two LCLs

estab-lished from Chinese wild-type EBV (CBC-SEB-CHI-3 and

-7)thatcarryunmethylatedgenomes: thetypeIBL cell line Rael that contains highly methylated EBV DNA and the

virus-producing B95-8 line. The materials and their EBV

antigen expression are listed in Table 1. The biopsy DNA

was cleaved in parallel with HpaII (methylation sensitive)

and MspI (methylation-insensitive isoschizomer) and

hy-bridized with the XhoIprobe (Fig. 1), coveringthe promoter

co

uM)

MH

4,0

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-2,0

-1,6

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00Ul

9

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1,0-0,50

0,39 -0,34

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-0,22

-(0,177)

-(0,146)

-(0,107)

-'V.

0

0

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Co

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af)

00

m Co

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M. H MW H M H M H M H

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6

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FIG. 6. Methylation of the oriP region in NPC biopsies. Same blot shown in Fig. 3 and 4,strippedandreprobed with the oriP probe (the 916-bp subfragmentdepicted inFig. la). The position of theMspI-MspI916-bp fragment in the control cell line B95-8 is indicated. Sample DNAs werecleaved withHpaII andMspI.

and all three coding exons of BNLF-I and extending over most of the 3'-untranslated region of the LMP-coding

tran-script. The B95-8 DNA showed a largely unmethylated

pattern (Fig. 3, lane B95-8 H) with four major fragments corresponding to the expected lengths of 818, 494, 357, and 253 bp from the sequence data which was comparable with

thefragments obtained withMspI. Therewas also one large

fragmentof around 2.5 kb, suggesting that some genomes in

B95-8 cells were completely methylated over the whole

region covered by the probe. Rael cells, in contrast,

con-tainedonly four large fragments (2.5, 3.0, and 4.0 kb and a

larger one). This reflects a high degree of methylation,

extending to CpG sites outside the region covered by the probe. The four smaller fragments were only seen in the

MspI (M) lane, confirming the absence of any detectable

hypomethylation. TheRael counterpart of the 494-bp B95-8

fragmentwas smaller, about 400 bp. The LCLs

CBC-SEB-CHI-3 and -7carried totallyunmethylated viral DNA in the

whole region. An 818-bp fragment was consistently present, but there was some size variation in the three smaller

fragments. CBC-SEB-CHI-3 had a Rael-like fragment size

distribution, while CBC-SEB-CHI-7 showedfragment sizes

intermediatebetween those of Rael and B95-8 cells. The size

variation of the 357- and 293-bp fragments may conceivably

influence the size of the LMP, since both fragments are

located within the majorBNLF-1 coding exonof LMP. Incontrast toB95-8cells and thetwocordblood-derived

cells, all the NPC tumors showed extensive EBV DNA

methylation.Nudemouse-passaged CAO andbiopsytumors

4, 5, and 10 contained one major unmethylated 357-bp

fragment, corresponding tothe BNLF-1 codingexon. CAO

andtumor4expressedLMP, whiletumors5and 10didnot.

The unmethylated fragment gave a weak signal in three

cases, suggestingthat itwasonlypresentinafraction of the EBV genomes. In biopsy tumor10 the fragment was

equi-molar with the otherfragments. These fourtumors and the

remaining seven showed the presence of much larger

frag-ments, which weretheresultofincompletecleavage due to

methylation protection of restriction sites. The

resulting

fragmentsvaried between0.7and5kb.Mostof themwerein

the size order of 1.5 to 4 kb. They all gave strong signals.

This suggests that theHpaII restriction sitesaremethylated

and thatmethylation extends fromafew hundred base

pairs

to 1kboutside theprobedregion, in most cases. Atleast10

of the tumors showed traces of uncleaved DNA. These

fragments have beenderived from genomes thatwere

meth-ylated over several tens of kilobases outside the BNLF-1 region. This is also supported by the lack of any incomplete cleavage with MspI. Moreover, staining the gels with ethid-ium bromide before blotting showed that good digestion occurred (data notshown). All NPC biopsies contained the 818-bpfragmentafter MspI cleavage; the smaller fragments were of various sizes, as in the cell lines. The 357-bp fragmentwasnotdetectable ineightof the tumors;onlytwo

smallfragments could be seen. Seven tumors contained the

400-bp fragment associated with Rael cells and lacked the

494-bp fragmentassociated with the B95-8 virus.

The LMP-coding exons varied slightly in size between different biopsies.Theywerehighlymethylated in the whole coding region, both in LMP-positive and LMP-negative

tumors. In contrast, agood correlation wasfound between

themethylation statusof the5'-flanking region of the LMP

geneanditsexpression.Theflanking regionwascompletely

unmethylatedinseventumors (tumors6, 12, 2,4, 13,8, and

C15), all of which expressed LMP (Fig. 4). The flanking

region was partially methylated in CAO, which expressed

LMPat alow level. Theregionwasextensively methylated

in tumors 3, 5, and 10, which did not express LMP. The smallestfragmentthat could be obtainedfrom thesetumors

after digestion with HpaII was2.5 kb. Similar tests on the

various control cell lines further corroborated the close

relationship between methylation of the 5'-flanking region

and lackofexpression, orviceversa. The twohumancord blood-derived LCLs and the B95-8 line expressed LMP,

whereas theRael line didnot. TheLMP-flanking regionwas

unmethylated in theformer three but highly

methylated

in

Rael, asexpected.

The5'-flankingregion oftheLMPgene showed

consider-ablemicroheterogeneitybetween thedifferent virus isolates.

Thelarge,506bpB95-8fragmenthad400-bpcounterparts in

nineof thetumorsandin the cord blood-derived cells.Itwas larger in biopsy 8 and of the same size in Rael. The resolution of the smaller fragments was not sufficient to detect minor variations.The

175-bp

fragment

was about the

same size inallviralDNAsexamined.

Three ofthree LMP-negative

biopsies

showed extensive

methylationof the whole800-bp

region

immediately

5' of the

BNLF-1promoterand

extending

outside into theLMPgene.

The same region was

completely

unmethylated

in six of

seven LMP-positive tumors. The seventh

(CAO)

was

par-tially methylated around the

large

400-bp

fragment

but

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0

M u MH A H u H

6 6

m

mi

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UN C)

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1i H M H Mi H

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am

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a.

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FIG. 7. Methylation of the BamHI K fragmentandEBNA-1exoninNPCbiopsies. Southern blotshowingNPCbiopsiesand controlcell linesprobed with the whole BamHl K fragment (Fig. ld). Sample DNAs cleavedwithHpaII (H)orMspI(M).Thepositionsofmolecular

weight marker fragmentsareindicated, and themajor B95-8 fragmentscaneasilybe identified(1,132, 774,and529bp).All the NPCsamples show extensivemethylation of all fragments. They allexpressEBNA-1.The control cell lines CBC-SEB-CHI-3 and -7gaveveryweaksignals with thisexposure,although thesameamountof cellular DNAwasloadedinalllanes. The BamHI Kfragmentwasunmethylatedin these

samples.

yielded one or twosmaller fragments, because ofa lack of

methylation of the surroundingrestriction sites.

Figure 5 is a schematic restriction map of the relevant BNLF-1 region and its 5'-flanking sequence in an

LMP-positive (tumor 12) andanLMP-negative (tumor 10) biopsy.

The dashedlines indicate the size variations of these frag-mentscompared with those of B95-8 cells.

Lack of methylationin the oriP region. A916-bp probewas used to analyze methylation in the oriP region. The probe

covers the middle part of the region, with the EBNA-1

enhancerjustupstreamandthe EBNA-1-binding dyad

sym-metry motifin the 3' end (31, 38).

Thesameunmethylated 916-bp fragmentwas detectedin 11tumorsand inthe controls (Fig.6). Inourprevious study

(12) the sameregion was unmethylated in all BL cells and LCLs examined and represented the only known area

ex-empted from high overall EBV DNA methylation in thetype

I BLline Rael.

TheexonofEBNA-1showedahigh degree ofmethylation in alltheNPCtumors (Fig. 7).

Methylationof theBamHI W enhancer.Recently,a

B-cell-specific enhancer in the BamHI W fragments that can influencetheexpressionof EBNA-2wasdescribed(33).It is contained withinanAvaIfragment(Fig. 1).Wescreenedthe DNA from nine NPCtumorsand controls with thisfragment

as alabeledprobeafter MspI andHpaII cleavage (Fig. 8).

All were EBNA-2 negative by immunoblotting. All were

methylatedin this region, althoughnotasextensively as in the LMPregion. Thus,awhole ladder offragments, ranging from 0.9to4 kb, was generated afterHpaII cleavage. The

single 558-bp fragmentfound inunmethylatedDNAwasnot detected inanyofthem,however.AfterMspI cleavage,this

fragment was detected in all tumors. It was slightly larger than that inB95-8cells,around 600bp.As shownearlier,the EBNA-2 exon was methylated in NPC tumors (12). The BamHI W enhancerregion was unmethylated in the LCLs thatexpressedEBNA-2 (Fig. 8).

DISCUSSION

We havefoundan overalldifference in EBV DNA

meth-ylation of NPC tumors and EBV-transformed LCLs of co

u H co U)

U H A H

a.

.s'll

.1

..i;...:...

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Cl XC.

to T- CJ s

-M H M H M H M H M H

C.,)

I

C-)

CD (0)

6

Ca C.)

M H M H

m

C-)

cn

CDO

6

o

m 4: 0 C.)

MN M H

UHM

M HM H MNH

lo

p.

I&

a,

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.0J6

FIG. 8. Methylation of theBamHI W enhancer. The same blot as in Fig. 3, 4, and 6, stripped and reprobed with an AvaI-AvaI probe covering the BamHI W enhancer (Fig. lb). The DNA samples were cleaved with HpaIl or Mspl. The control cell lines B95-8, CBC-SEB-CHI-3, and -7 expressed EBNA-2 to -6, while all the other samples (NPC biopsies) expressedonly EBNA-1. The 558-bp fragment could easilybeidentified in the control cell line B95-8, and it is slightly larger in all the other samples.

normal B-cell origin. EBV genomes are extensively methyl-ated in theformer and unmethylated in the latter. We have foundasimilar difference previously between the type I BL line Rael and the derived LCLs (12).

In addition to this overall difference, there is a superim-posed, expression-related difference in the 5'-flanking re-gions which is known or suspected to contain regulatory sequences (Table 2).

It is known that methylation of 5'-flanking regulatory sequences mayinfluence gene expression, whereas

methyl-ation ofthecoding exonshas no such effectas arule (3, 11,

25, 26). The recent findings of single, specific methylation sites within the DNA binding motif of several proteins

involvedintranscriptionalregulation show a potential direct

role ofmethylation in gene control (2, 26, 37, 41). A search

for specific critical CpG methylations within the relevant

promoter and enhancer regions in the EBV genome will

obviouslybe of great interest. Our data suggest that

meth-ylation of regulatory regions influences EBNA and LMP control.

The promoter for LMP-coding transcripts has been mapped to the EDL-1 site (coordinate 169546). We have foundacomplete correlation between LMPexpressionand lackofmethylation of the promoter region andsome 800bp upstream of it. This is particularly remarkable since the surrounding EBV DNA is extensively methylated in NPC tumors,includingthe threeLMP-encodingexons. Thesame

region was methylated in all tumors that failed to express

LMP. This suggests that methylation participates in the

regulationof LMPexpression. Direct studies onLMP

tran-scriptional regulation will be necessary to establish any

direct causalrelationship.Theregionthatshowedan expres-sion-correlated methylation pattern coincides with the

re-cently described LMPregulatorysequence (17).

Promotersfor EBNAtranscripts havebeen found in two

[image:8.612.139.474.76.332.2]

regions so far, BamHI-C (BCR 2, P11305) and BamHI-Ws

TABLE 2. DNAmethylationand geneexpressionpatterns incodingand control regions

Methylation' Proteinexpression Celltype

oriP EBNA-1 exon BamHI Wenhancer EBNA-2exon BNLF-15'sequence LMPexon EBNA-1 EBNA-2 LMP

NPC(LMP+) - + + + - + + - +

NPC(LMP-) - + + + + + + -

-C15 - + + + + + +

C15 LCL - - - + + +

Rael - + + + + + +

CBM1-Ral-STO - - - + + +

aoriP, Originof latent DNAreplication; EBNA-1exon, BKRF-1 exonfor EBNA-1 protein;BamHI Wenhancer, enhancer5'ofWpininternalrepeat1;

EBNA-2 exon, BYRF-1 codingexonforEBNA-2; BNLF-1 5' sequence, LMPcontrol region;LMP exon, threeLMP-codingexons. +,Methylation; -, no

methylation.

co 0) m

a

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M H M H

U: a

4,0

-3,0

-2,0

-1,6

-1,0

-0,50

-O

S.

so 1

II

_.

..1

0lo"m

X,F.

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[image:8.612.54.557.609.699.2]
(9)

TABLE 3. NPCbiopsy protein expressiona

No.expressing:

No.of biopsies Reference EBNA-1 LMP

24 24 9 43

56b 56 36 14

13 13 10 Thispaper

aProtein expressionwastestedbyimmunoblottingwithpolyvalent and/or

monospecific reagents ofEBNA-1 and LMP. EBNA-2 to -6 werealways

negative.

bOnlyEBNA-1-and/orEBVDNA-positivebiopsiesareincluded.

(P14352+nx30739

n = 4 to 11). In the producer line B95-8,

several different EBNA transcripts start at the same pro-moter,P11305,whereas EBNA-2transcriptsinanLCL,IB4,

werefound toinitiate at one of the BamHI W promoters. An enhancer that can transactivate after EBNA-1 binding is

localized in oriP 3 kb upstream of the BamHI C promoter (31). The oriP enhancer may be involved in regulation of transcripts from the BamHI C promoter (38).

The oriP region has been unmethylated in all EBV-carrying tissues studiedsofar, includingBL andNPC.This

mayfitwell with thefactthat EBNA-1is the mostregularly

expressed EBVprotein, is found in all EBV DNA-carrying

cells studiedsofar,andisunaffectedbythe cell

phenotype-dependentexpression and regulation.

Recently, it was demonstrated that in a selection of

BL-derived cell lines and invitro-transformed LCLs,either the BamHI W or the BamHI C promoterwasused

irrespec-tive of cell phenotype (42). This study, however, did not

includeanytype I BLcell linesorNPCtumorbiopsies.The

EBNA-1 transcription initiation remains to be identified in these cell types. Hittetal.(22)detected notranscriptsfrom the BamHI W region when screening their cDNA library

derivedfromthenudemouse-passaged NPCC15,suggesting

that EBNAexpressioninthisNPCiscontrolled inadifferent way and without the extensive splicing of the BamHI W

regionsthat has beenseeninB-celllines (Raji, B95-8, IB4,

andJY).

The lack ofmethylation oforiPmay also besecondaryto

its involvementin DNAreplication or may evenresultfrom

theprotection by DNA-binding regulatory proteins,

includ-ing EBNA-1. Recently, an enhancer that is 50 to 400 bp upstreamof the BamHI Wpromoter(33) and thatfunctions

in B-cell lines but not in HeLa cells has also been demon-strated.This enhancer is likely to beimportant for regulation of EBNA expression. The BamHI W promoter-enhancer

was hypomethylated in the EBNA-2-expressing LCLs but

methylated in all the nonexpressing NPCs.

Our study has further confirmed the specific pattern of

EBVprotein expression in NPC: EBNA-1 expressionin all tumors that are EBV DNA positive, and LMP expression

only in a large fraction. This fraction has been repeatedly

higherin Chinese NPCs than in NPC materials from other

high or medium-high endemic regions. Earlier we detected LMP in 71% of Chinese tumors (12), and now we have

detected LMPin 10 of 13 (77%) tumors in two consecutive,

randomly chosen groups (Table 3). This compares with 25 to

62% in earlier investigated tumors from North Africa and

EastAfrica (14, 43).

LMP can transform established strains of rodent

fibro-blasts (1, 40).

F?ahraeus

et al. (15) have recently found that

expression of LMP in an EBV-negative epithelial cell line

(RHEK-1) changes the morphology of the transfected cells

andinhibits keratinization. Dawsonet al. (7) demonstrated

simultaneously

that LMP inhibitsdifferentiation inahuman keratinocyte cell line. NPC cells are low differentiated or

anaplastic.Whethertheexpressionof LMPcanberelatedto

the level ofdifferentiation andtothe clinical course of the

tumorremainsa question.

ACKNOWLEDGMENTS

Weare grateful toDavid Thorley-Lawson and Martin Rowe for providing LMP MAbs, to Georg Bornkamm for providing EBV DNAclones, toMaria GraziaMasuccifor fruitful discussions,and to Eva Ragnar for technicalassistance.

This study was supported by the Swedish Cancer Society, the SwedishMedical ResearchCouncil (project 5667),andPHSgrant5 RO1CA28380-03 from theNational CancerInstitute. H.L.-F. and J.M. are the recipients of visiting scientist fellowships from the Swedish CancerSociety. H.L.-F. is alsosupportedbytheNational NatureScience Foundation andNationalOncogeneResearch Cen-ter in China.

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38. Sugden, B., andN. Warren. 1989. ApromoterofEpstein-Barr virus that canfunctionduringlatentinfection can be transacti-vated by EBNA-1, a viral protein required for viral DNA replication duringlatentinfection. J. Virol.63:2644-2649. 39. Tugwood, J., W.-H. Lau, S.-K.0,S.-Y.Tsao,W. M.C.Martin,

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40. Wang,D.,D.Liebowitz, and E. Kieff. 1985. An EBV-membrane proteinexpressed inimmortalized lymphocytes transforms

es-tablished rodent cells.Cell 43:831-840.

41. Watt, F., and P. L.Molloy.1988.Cytosinemethylationprevents bindingtoDNAofaHeLacelltranscriptionfactorrequiredfor optimal expression of the adenovirus major late promoter. GenesDev. 2:1136-1143.

42. Woisetschlaeger, M., J.L. Strominger, andS.H. Speck. 1989. Mutually exclusiveuseof viral promoters inEpstein-Barrvirus latently infected lymphocytes. Proc. Natl. Acad. Sci. USA 86:6498-6502.

43. Young,L.S., C.W.Dawson,D.Clark,H.Rupani,P.Busson,T.

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Figure

FIG.1.BamHIrestrictionblotting.(e.g.,byfragment,fragmentprobeslatterresultingregionslatentbraneMspl-MspIenhancerTwoEBNA-2,regionrecognizingwithinasTheEBNA-2-codingandcleaved an DNA probes used to study methylation by Southern Horizontal black lines (_) bet
FIG. 2.Ramosas10serum, the were Immunoblots showing expression of EBV proteins in NPC biopsies
TABLE 1. Designations and EBV protein expression in biopsiesand cell lines used in this study
FIG. 5.areonesequencesLMP Schematic drawing of the BNLF-1 region and upstream in two NPC biopsies (one expressing LMP [b; no
+4

References

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